1. Field of the Invention
The field of the invention is methods and devices for the growth of normal mammalian cells in culture, including the maintenance and selective growth of human stem and/or hematopoietic cells.
2. Discussion of the Background
There is significant interest in the ability to use cells for a wide variety of therapeutic purposes. The hematopoietic system exemplifies the extraordinary range of cells involved in protection of mammalian hosts from pathogens, toxins, neoplastic cells, and other diseases. The hematopoietic system is believed to evolve from a single stem cell, from which all the lineages of the hematopoietic system derive. The particular manner in which the stem cell proliferates and differentiates to become determined in its lineage is not completely understood, nor are the factors defined. However, once the stem cell has become dedicated to a particular lineage, there appear to be a number of factors, for example colony stimulating factors, which allow, and may direct the stem cell to a particular mature cell lineage.
There are many uses for blood cells. Platelets find use in protection against hemorrhagings an well an a source of platelet derived growth factor. Red blood cells can find use in transfusions to support the transport of oxygen. Specific lymphocytes may find application in the treatment of various diseases, where the lymphocyte is specifically sensitized to an epitome of an antigen. Stem cells may be used for genetic therapy as well as for rescue from high dose cancer chemotherapy. These and many other purposes may be contemplated.
In order to provide these cells, it will be necessary to provide a means, whereby cells can be grown in culture and result in the desired mature cell, either prior to or after administration to a mammalian host. The hematopoietic cells are known to grow and mature to varying degrees in bone, as part of the bone marrow. It therefore becomes of interest to recreate a system which provides substantially the same environment as is encountered in the bone marrow, as well as being able to direct these cells which are grown in culture to a specific lineage.
In this vein, U.S. Pat. No. 4,721,096 describes a 3-dimensional system involving stromal cells for the growth of hematopoietic cells. See also the references cited therein. Glanville et al., Nature (1981) 292: 267-269, describe the mouse metallothionein-I gene. Wong et al., Science (1985) 228: 810-815, describe human GM-CSF. Lemischka et al., Cell (1986) 45: 917-927, describe retrovirus-mediated gene transfer as a marker for hematopoietic stem cells and the tracking of the fate of these cells after transplantation. Yang et al., Cell (1986) 47: 3-10, describe human IL-3. Chen et al, Okayama, Mol. Cell. Biol. (1987) 7: 2745-2752, describe transformation of mammalian cells by plasmid DNA. Greaves et al., Cell (1989) 56: 979-986, describe the human CD2 gene. Civin et al, J. Immunol. (1984) 133: 1576-165, describe the CD34 antigen. Martin et al., Cell (1990) 63: 203211, describe human S-CSF. Forrester et al, J. Cell Science, (1984) 70: 93-110, discuss a parallel flow chamber. Coulombel et al., J. Clin. Invest., (1986) 75: 961, describe the loss of CML cells in static cultures.
Tissue Engineering is a new and growing part of biotechnology. Its goal is to reconstitute fully or partially functioning human tissue in vitro to enable a variety of clinical and other applications. Several studies have been carried out recently that are aimed at reconstituting functioning human tissues in vitro. To date, perhaps the cultivation of human skin has been most successful.
The development of prolific in vitro human bone marrow systems has been long desired since such systems would enable a broad range of clinical, as well as scientific, applications. Such applications include:
(1) study of the basic dynamics of hematopoietic differentiation,
(2) improved autologous and allogeneic bone marrow transplantation,
(3) depletion of undesirable cells upon bone marrow transplantation, such as T-cells or any malignant cells,
(4) gene therapy of the blood cell system, and
(5) the large-scale production of mature blood cells, such as red cells and platelets.
Although long-term human bone marrow cultures (LTHBMCs) developed in the late 1970s and early 1980s were initially disappointing in their longevity and cell productivity (see Greenberger (1984) "Long-term Hematopoietic Cultures," pp. 203-242 in "Hematopoiesis", D. W. Golde, Editor, Churchill-Livingstone, N.Y.), recent advances have markedly improved their performance. However, these improvements are carried out with a sub-clinical number of bone marrow cells in standard laboratory size tissue culture hardware. Therefore, a compelling and profound need exists for providing methods, compositions and devices that can carry a clinically meaningful number of human bone marrow cells to enable the therapies and applications described above.
These recent improvements in LTHBMC performance have used in vivo simulation in an attempt to create culture conditions that are conducive to in vitro reconstitution of hematopoietic function. A series of studies have demonstrated that this approach is successful. The function of the supporting stromal cell layer (mostly fibroblast, with some adipocytes and endothelial cells) has been shown to be significantly influenced by the medium perfusion rate, or the medium exchange schedule. Metabolic function, growth, and perhaps most importantly growth factor secretion have all been shown to be influenced by the medium exchange rate for normal human bone marrow fibroblasts (Caldwell et al, J. Cell. Physiol., (1990) 147: 344-353), and even for transfected NIH-3T3 murine cells (Caldwell et al, Biotech. Prog., (1991) 7: 1-8).
The ability of stroma to support human hematopoiesis in vitro has been demonstrated by the inventors to be enhanced by rapid medium exchange. See Schwartz et al, Proc. Nat. Acad. Sci. (USA), (1991), 88: 6760-6764 or U.S. patent application Ser. No. 07/737,024, filed Jul. 29, 1991, now abandoned. Under rapid medium exchange and at high cell densities, LTHBMCs can support the stable production of progenitor cells up to 20 weeks in culture and prolong granulopoiesis up to 19 weeks. The former result is notable in that it shows that these culture conditions can provide conditions suitable for stem cell maintenance and proliferation in vitro for extended periods of time.
Judicious use of added soluble growth factors can further improve the performance of these cultures. Some hematopoietic growth factors, such as Interleukin-3 (IL-3) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), are believed to stimulate the differentiation of early hematopoietic cells. Other growth factors, such as Erythropoietin (Epo), are believed to be terminal differentiation factors that stimulate the production of mature cells of a particular lineage. It has been observed that the addition of soluble IL-3, GM-CSF and Epo in rapidly perfused human bone marrow cultures can significantly stimulate the production of mature and progenitor cells for periods of up to 6-8 weeks. During this period the cell culture regeneration rate (the time it takes to produce as many non-adherent cells as initially seeded) is about 2 weeks (for comparison, the estimated in vivo rate is about 2 days) and erythropoiesis is observed throughout the 20 week culture period. Both results are remarkable since all previous attempts to expand human bone marrow in vitro have proved unsuccessful and erythropoiesis is short lived in traditional LTHBMCs (lasting less than 2 weeks).
Thus, adjustment of culture conditions to simulate the in vivo condition more closely has dramatically improved the progenitor and non-adherent cell productivity of LTHBMCs. Further, these conditions lead to the reconstitution of blood cell lineages other than the macrophagic lineage which has been observed to dominate the composition of the non-adherent cell population in LTHBMCs and bone marrow cultures from other animal species (see review in R. M. Schwartz, "Optimization of Long-Term Bone Marrow Cultures," PhD thesis, 1991, University of Michigan). Further supplementation of the medium with the stem-cell factor (SCF, also known as the c-kit ligand of the mast cell growth factor) and interleukins 1 and 6 lead to even greater expansion in cell numbers. To date, this composition has not been publicly disclosed.
The discovery of prolific conditions for long-term maintenance and proliferation of early human hematopoietic cells in vitro in small scale standard cell culture laboratory hardware is clearly important. Even more important is the development of methods, devices and compositions that allow for the maintenance and proliferation of these cells in clinically meaningful numbers so that the important therapeutic applications, described above, can be carried out.
Bioreactor designs, which address the question of harvesting cells produced in the bioreactor, have been proposed. Interestingly, these proposed designs provide only for batch-wise harvesting of the cells by opening the reactor once a sufficient number of cells is obtained, thereby stopping the culture. For example, U.S. Pat. No. 5,010,014 describes a cell culture chamber unit comprising a cell culture region and a gas region separated by a gas-permeable wall which permits batch-wise cellular harvesting. U.S. Pat. No. 4,839,292 describes a cell culture flask which comprises two chambers separated by a gas permeable membrane. Each chamber is described as being equipped with both inlet and outlet means, and the flask is described as being suitable for batch-wise harvesting of the cells by removal of the gas permeable membrane from the reactor.
U.S. Pat. No. 4,948,728 describes a bioreactor and the use of a membrane comprised of a ceramic layer and a hydrophobic layer, with a biofilm attached to the ceramic film. This patent however does not address the question of cell harvesting.
Further, there is a need for a bioreactor permitting the maintenance of a balanced (in terms of cell type) complex primary cell culture. Human stem or hematopoietic cell cultures are very sensitive to their dynamic (i.e., rates of gas/nutrients/growth factor supply and removal) and chemical environment. Today no bioreactor design satisfactorily permits such maintenance of a balanced complex primary cell culture.
Available designs accordingly do not provide a method for harvesting cells without disrupting the culture or the maintenance of a balanced complex primary cell culture, much less both. A suitable design is thus needed permitting the maintenance and proliferation of human stem cells and/or early human hematopoietic cells in vitro, and advantageously further permitting harvesting cells produced in the reactor without disrupting the culture. There is a strongly felt need for such a design.